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BIOLOGY

BIOTECHNOLOGY
PRINCIPLES AND
PROCESSES
ONAM EXAM SPECIAL
 BIOTECHNOLOGY : Recombinant DNA
PRINCIPLES AND PROCESSES A recombinant DNA [rDNA] is an artificial DNA
constructed by combining DNA from two dif-
ferent Organisms.

Construction of the first

Definition (by EFB) recombinant DNA
The integration of natural science and organ-
isms, cells, parts and molecular analogues for COHEN AND BOYER'S EXPERIMENT
products and services’.
 The construction of the first recombinant
DNA is done by linking antibiotic resistance
gene with a plasmid of salmonella typh-
imurium.
 Stanley Cohen and Herbert Boyer accom-
plished this in 1972.

Basic steps in genetically modify-

ing an organism
Principles of Biotechnology
 Identification of DNA with desirable genes
GENETIC ENGINEERING  Introduction of the identified DNA into the
It is the technique to alter the genetic host
material (DNA and RNA), and ton introduce
these into the host organism, thus changing  Maintenance of introduced DNA in the host
the phenotype of the host organism. and transfer of the DNA to its progeny

BIOPROCESS(CHEMICAL) ENGINEERING
It involves the manufacture of biotechnolog- TOOLS OF RECOMBINANT DNA
ical products like antibiotics, vaccines, en-
zymes, etc. TECHNOLOGY
 Restriction enzymes
THE TECHNIQUES OF GENETIC
ENGINEERING INCLUDE;  Polymerase enzymes
 Creation of recombinant DNA  DNA ligases
 Gene cloning  Cloning vector
 Gene transfer  Host organism

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Restriction enzymes
 These are enzymes which cut DNA into
fragments at specific sites. These are com-
monly called as "molecular scissors".
 Two kinds; exonucleases and endonucle-
ases
 Exonucleases remove nucleotides from the
ends of the DNA whereas, endonucleases
make cuts at specific positions within the
DNA.
 The first discovered restriction endonucle-
ase is Hind II.
 Hind II always cut DNA at a particular point
by recognising a specific sequence of six
base pairs. This specific base sequence is The overhanging projections are called sticky
known as the recognition sequence. ends. These are named so because they form
hydrogen bonds with their complementary cut
counterparts.

NOMENCLATURE OF RESTRICTION ENZYMES

EcoRI comes from Escherichia coli RY 13. In
Sticky and Blunt
ECORI, the letter ' R ' is derived from the name Ends
of strain. The Roman numbers following the
names indicate the order in which the en-
zymes were isolated from that strain of bac-
teria.
PALINDROMIC NUCLEOTIDE SEQUENCES
The palindrome in DNA is a sequence of base
pairs that reads same on the two strands
when orientation of reading is kept the same.

Palindromic nucleotide DNA Ligases

sequences These are enzymes which helps to join DNA
fragments.

MALAYALAM: a word palindrome

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DNA Polymerases (I) ORIGIN OF REPLICATION (ORI) :
This is the sequence where replication
These are enzymes which helps in the starts.
replication of DNA.
(II) SELECTABLE MARKER :
 These are markers used to identify a host
organism.
 Normally, the genes encoding resistance
to antibiotics such as ampicillin, chloram-
phenicol, tetracycline or kanamycin, etc.,
are considered as useful selectable markers
for E. coli.

Cloning Vectors
These are vehicles used to carry genes
from one cell to another cell or from one or-
ganism to another organism.

Bacteriophages because of their high number

per cell, have very high copy numbers of their

genome within the bacterial cells.
Some plasmids may have only one or two
(III) CLONING SITES:
copies per cell whereas others may have 15-
In order to link the foreign (alien) DNA, the
100 copies per cell.
vector needs single, recognition sites for the
commonly used restriction enzymes.

Characteristic features
of a vector
 Origin of replication (ori)
 Selectable marker
 Cloning Sites

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 E. coli cloning vector pBR322 showing restric-
Methods to identify transfor-
tion sites (Hind III, EcoR I, BamH I, Sal I, Pvu
II, Pst I, Cla I), ori and antibiotic resistance
mants & recombinants
genes (ampR and tet tR ). Rop codes for the
proteins involved in the replication of the SELECTABLE MARKER
plasmid.

(IV) VECTORS FOR CLONING GENES IN PLANTS

AND ANIMALS
 Agrobacterium tumifaciens, a pathogen
of several dicot plants is able to deliver a
piece of DNA known as ‘T-DNA’ to trans-
form normal plant cells into tumor
cells.
 The tumor inducing (Ti) plasmid of Agro-
bacterium tumifaciens has now been
modified into a cloning vector which is Colour Reaction
no more pathogenic to the plants but is
still able to use the mechanisms to de-
liver genes of our interest into a variety of
plants.
 Retroviruses have also been disarmed
and are now used to deliver desirable
genes into animal cells.

Transformation
Transformation is a procedurethrough which a  The presence of a chromogenic substrate
piece of DNA is introduced into a host bacteri- gives blue coloured colonies if the plasmid
um. in the bacteria does not have an insert.
Transformants Non-transformants  Presence of insert results into insertional
Recombinants Non recombinants inactivation of β - galactosidase and the
colonies do not produce any colour, and
are identified as recombinant colonies.

Selectable Marker
Competent Host
 In this method, one antibiotic resistance
gene (ampR)helps in the selectionof trans-
A host organism which is capable of taking
formants, whereas theother antibiotic resis-
up foreign DNA is called a competent Host.
tance gene (tetR) gets ‘inactivated due to
insertion’ of foreign DNA, and helps in selec-
tion ofrecombinants. Space for Keynotes

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Methods to make Disarmed pathogen vectors
competent host
 Pathogenicity of vector is removed- ‘dis-
(i) This is done by treating them with a spe- armed pathogens’.
cific concentration of a divalent cation, such  Disarmed pathogen vector - allowed to in-
as calcium. fect host, and transfer rDNA.
(ii) Recombinant DNA can be forced into such
cells by incubating the cells with recombi-
nant DNA on ice, followed by placing them
briefly at 42 C (heat shock), and then putting
them back on ice.
PROCESSES OF RECOMBINANT DNA
Methods of Gene TECHNOLOGY
transfer 1. Isolation of DNA
2. Fragmentation of DNA
MICRO-INJECTION
3. Separation of DNA
4. Ligation of DNA
5. Culturing of DNA

Common for animal cell.

 rDNA directly injected into nucleus of host
cell.

Biolistics or Gene gun

Space for Keynotes

Used for plant cell.
 Host cell bombarded with high velocity mi-
cro-particles of gold or tungsten.
 Micro-particles coated with recombinant
DNA.

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Isolation of DNA Separation and isolation of DNA
 This can be achieved by treating the bac-
terial cells or plant or animal tissue with en-
zymes such as lysozyme (bacteria), cellu-
lase (plant cells) &chitinase (fungus).
 The RNA can be removed by treatment with
ribonuclease whereas proteins can be re-
moved by treatment with protease.
 Other molecules can be removed by ap-
propriate treatments and purified DNA ul-
timately precipitates out after the addition
of chilled ethanol.
 DNA that separates out are removed by  The fragments can be separated by a
spooling technique known as gel electrophoresis.
 Since DNA fragments are negatively
charged they can be separated by forcing
them to move towards the anode under an
Isolation of DNA electric field through a medium.

Bacteria Lysozyme
Plant cells Cellulase
Fungus Chitinase The most commonly used matrix is agarose
Protein Protease which is a natural polymer
RNA Ribonuclease extracted from sea weeds.
 The DNA fragments separated according
to their size through sieving effect provided
by the agarose gel. Hence, the smaller the
DNA Spooling fragment size, the farther it moves.
 The separated DNA fragments can be visu-
alised only after staining the DNA with a
compound known as ethidium b r o m i d e
followed by exposure to UV radiation.
 The separated bands of DNA are cut out
from the agarose gel and extracted from
the gel piece. This step is known as elu-
tion.

Space for Keynotes

Fragmentation of DNA
Restriction enzyme digestions are performed
by incubating purified DNA molecules with the
restriction enzyme, at the optimal conditions
for that specific enzyme.

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Amplification of Gene of Interest Bioreactors

 PCR stands for Polymerase Chain

Reaction. Bioreactors are vessels in which raw
materials are biologically converted into spe-
 The enzyme DNA polymerase extends the cific products, individual enzymes, etc., using
primers using the nucleotides provided in microbial plant, animal or human cells.
the reaction and the genomic DNA as tem-
plate. SIGNIFICANCE OF A BIOREACTOR
A bioreactor provides the optimal conditions
 If the process of replication of DNA is re-
for achieving the desired product.
peated many times, the segment of DNA
can be amplified to approximately billion
times. TYPES OF BIOREACTORS
 Such repeated amplification is achieved by
the use of a thermostable DNA polymerase
(isolated from a bacterium, Thermus
aquaticus), which remain active during the
high temperature induced denaturation of
double stranded DNA. Simple stirred-tank bioreactor Sparged stirred-tank bioreactor

Ligation of DNA
The amplified DNA fragment are ligated with
a suitable vector for cloning.
TRANSFERRING THE RECOMBINANT DNA
INTO THE HOST CELL COMPONENTS OF A BIOREACTOR
The rDNA are introduced into the host
cells by direct and indirect methods.  Agitator system
 An oxygen delivery system
 A foam control system
Culturing the host cells in a medium at large  A temperature control system pH control
scale and extraction of the desired product system
If any protein encoding gene is expressed in
 sampling ports so that small volumes of
a heterologous host, it is called a recombi-
the culture can be withdrawn periodically.
nant protein.

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 DOWNSTREAM PROCESSING
The recombinant protein product has
to be subjected through a series of pro-
cesses before it is ready for marketing as a
finished product.
THE PROCESSES INCLUDE
 separation
 purification,
which are collectively referred to as
downstream processing

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